This invention relates generally to molecular biology, and more particularly, to a new method allowing coupling of the transcription of RNA from a template DNA and the translation of the RNA in eukaryotic cellular lysates or other extracts.
The steps involved in the transcription and translation (expression) of genes in cells are very complex and are not yet completely understood. There is a basic pattern that must be followed, however, for protein to be produced from DNA. The DNA is first transcribed into RNA, and then the RNA is translated by the interaction of various cellular components into protein. In prokaryotic cells (bacteria) transcription and translation are "coupled", meaning that RNA is translated into protein during the time that it is being transcribed from the DNA. In eukaryotic cells (animals, plants) the two activities are separate, making the overall process much more complicated. DNA is transcribed into RNA inside the nucleus of the cell, but the RNA is further processed into mRNA and then transported outside the nucleus to the cytoplasm where it is translated into protein.
The ability of molecular biologists to isolate and clone genes, as well as their ability to isolate particular mRNAs or "messages" from cells, has brought about the need for systems which can be used to express these genes or messages. The expression of a gene is important in the overall understanding of its function and regulation. Methods are now available for rapid expression of proteins, making it possible to manipulate genes and then study the effect of the manipulations on their function. The amount of protein to be produced, whether the gene is prokaryotic or eukaryotic and the relative merits of an in vitro cell-free or an in vitro whole-cell system, are some of the factors considered by researchers when selecting an expression system. The choice of a system is influenced by the gene being studied. For the most part, a prokaryotic gene is expressed best in a prokaryotic system, and a eukaryotic gene is more efficiently and accurately expressed in a eukaryotic system. This is because of the many regulatory sequences and promoters that are recognized more efficiently in a like system. The expression of genes can be achieved in both in vitro whole-cell and in vitro cell-free systems.
In vitro transcription systems using prokaryotic or eukaryotic cells are available, however, these systems are difficult to work with since intact cells are used. In vitro cell-free systems, on the other hand, are made from cell-free extracts produced from prokaryotic or eukaryotic cells that contain all the necessary components to translate DNA or RNA into protein. Cell-free extracts can be prepared from prokaryotic cells such as E. coli and from eukaryotic cells such as rabbit reticulocytes and wheat germ. Cell-free systems are very popular because there are standard protocols available for their preparation and because they are commercially available from a number of sources.
E. coli S30 cell-free extracts were first described by Zubay, G. (1973, Ann. Rev. Genet. Vol 7, p. 267). These can be used when the gene to be expressed has been cloned into a vector containing the appropriate prokaryotic regulatory sequences, such as a promoter and ribosome binding site. Prokaryotic E. coli cell-free systems are considered "coupled" because transcription and translation occur simultaneously after the addition of DNA to the extract. The use of RNA as a template in E. coli extracts results in protein production but such a reaction is not coupled. Rabbit reticulocyte lysates and wheat germ extracts are used preferably for the expression of eukaryotic genes or mRNA. Both systems require the use of RNA as the template for protein translation because, as previously mentioned, eukaryotic systems are not coupled.
Rabbit reticulocyte lysate was described by Pelham, H. R. B. and Jackson, R. J. (1976, Eur. J. Biochem. Vol. 67, p. 247). This expression system is probably the most widely used cell-free system for in vitro translation, and is used in the identification of mRNA species, the characterization of their products, the investigation of transcriptional and translational control. Processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes to a standard translation reaction (Walter, P. and Biobel, G. (1983) Meth. Enzymol. 96, 84). Rabbit reticulocyte lysate also contains a variety of post-translational processing activities, including acetylation, isoprenylation, proteolyis and some phosphorylation activity (Glass, C. A. and Pollard, K. M. (1990) Promega Notes 26).
Wheat germ extract was described by Roberts, B. E. and Paterson, B. M. (1973, Proc. Natl. Acad. Sci. USA, Vol. 70, P. 2330). Cell-free extracts of wheat germ support the translation in vitro of a wide variety of viral and other prokaryotic RNAs, as well as eukaryotic mRNAs. (Anderson, C., et al. (1983) Meth. Enzymol. 101, 635). Generally, it is found necessary to include a ribonuclease inhibitor in the reaction mix of a wheat germ translation system, as ribonuclease activities in wheat germ extract are present.
RNA for translational studies is obtained by either isolating mRNA or by making in vitro RNA transcripts from DNA that has been cloned into a vector containing an RNA polymerase promoter. The first method isolates mRNA or "message" directly from cells.
The second obtains RNA for in vitro translation by in vitro transcription. In vitro transcription of cloned DNA behind phage polymerase promoters was described by Krieg, P. and Melton, D (1984, Nucl. Acids Res., Vol. 12, p. 7057). This has become a standard method for obtaining RNA from cloned genes for use in in vitro translation reactions. This method requires that the DNA or gene of interest be cloned into a vector containing a promoter for one of the following RNA polymerases, SP6, T7 or T3. The vector is then linearized at the 3' end of the cloned gene using a restriction enzyme, followed by an in vitro transcription reaction to make RNA transcripts. A number of vectors containing the SP6, T7 and T3 RNA polymerase promoters are commercially available and are widely used for cloning DNA.
In any case, the process of obtaining RNA transcripts for use in rabbit reticulocyte lysate or wheat germ systems introduces a variable which can affect the efficiency of the translation reaction. Extra care must always be taken when working with RNA as it is easily degraded by ribonucleases. DNA templates are much more stable.
After rabbit reticulocyte lysate and wheat germ extract were developed as cell-free translation systems, attempts were made to couple transcription and translation. One system that was developed was a "linked" transcription and translation system (Roberts, B. E., et al. (1975), Proc. Natl. Acad. Sci. USA, Vol 72, 1922-1926). This system involved the use of wheat germ extract and looked at transcription and translation of SV40 viral DNA using E. coli RNA polymerase. In this system transcription occurs in 15 minute incubation step Just prior to the addition of the wheat germ extract. The steps are separated because of incompatibility between the buffer conditions necessary for transcription and those necessary for translation, and also because of the different temperature requirements for both processes. This system has a number of drawbacks. One is the lack of control over which protein product is produced, as a number of different proteins are synthesized simultaneously from the same SV40 DNA template. Although the authors of that study indicated that a coupled system had been developed no data for a coupled system was shown.
Another system was developed by Pelham, H. R. B, et al. (1978), Eur. J. Biochem., Vol. 82, 199-209, where coupled transcription and translation occurred after the introduction of vaccinia viral core particles into rabbit reticulocyte lysate. The production of vaccinia proteins from the viral DNA was presumably due to transcription by the endogenous vaccinia RNA polymerase and subsequent translation by the lysate. This system was limited by the fact that only vaccinia proteins would be produced while exogenous DNA from sources other than vaccinia would not be recognized by the RNA polymerase and therefore no transcription or translation could occur. Viral core particles had to be isolated, and the authors were unable to exclusively produce a single protein.
Work has also been described using "continuous" cell-free in vitro translation systems with the emphasis on large scale production of protein. Continuous systems are very different than the more common batch type, or static, in vitro cell-free translation reactions which occur in a contained reaction volume. Continuous translation involves a bioreactor (such as an Amicon 8MC ultrafiltration unit) in which large scale reactions are set up and protein is "continually" translated over extended periods of time. The reaction requires that a buffer be fed into the reaction as it progresses, and also requires that the products of translation be removed from the reaction filter unit. This type of system works well with E. coli S30 extract and wheat germ extract when RNA template is introduced. See Spirin, et al. (1988) Science, Vol 242, 1162-1164. The system also works using RNA templates in rabbit reticulocyte lysate. See Ryabova, et al. (1989) Nucl. Acid Res., Vol. 17, No. 11, 4412. The system is also known to work well with DNA templates in E. coli S30 extracts. See Baranov, et al. (1989) Gene, Vol 84, 463-466. PCT publication WO9102076 discloses continuous cell-free translation from DNA templates using eukaryotic lysates.
Continuous reactions are performed variously from tens of hours up to around one hundred hours, and require a substantial investment of time and resources to set up and run. Translation in a "continuous" system is also directed towards producing large amounts of protein, and differs substantially from standard (static) in vitro translation reactions. Static reactions can be run in a small reaction volume, typically measured in microliters, and are often completed in one to two hours. A static translation reaction is not directed towards producing preparative amounts (milligrams) of proteins. A static reaction is generally used to produce protein for investigative applications, such as the identification and characterization of mRNA species, or studies of transcriptional or translation control. None of the rabbit reticulocyte or wheat germ systems currently known for in vitro translation provide for coupled transcription and translation in a static reaction mixture.